Method of preparing an extract



for repeated administration to patients havconcentrating it with a reagent which will" unite with it as by adsorption, and separating said substance and rea ent.

3. A method for obtaining a potent substance from the ductlessportion of pancreatic or related glands in concentrated for repeated administration to patients hav ing the herein-described physiological char acteristicswhich consists 0 extractin said substance from the gland with a so vent, concentrating it with a reagent which will unite with it as by'ad'sorption, and making.

a sterile aqueous solution of the substance.

4- A methodfor obtaining a potent substance from the ductless portion of pancreatic or related glands in concentrated form and sufiiciently free from impurities for repeated administration to patients having the herein-described ph siological characteristics which consists 0 extracting saidw substance from the gland with asblvent, concentrating it with a reagent which will unite with it as by adsorption, separating the substance and the reagent, and making a sterile aqueous solution'of the substance.

5. A method for obtaining a potent substance from the ductless portion of pancreatic or related glands in concentrated form and suflicientlyfree from impurities for'repeated administrationto patients having theherein-described ph siological characteristics which consists o extracting said substance from the gland with a solvent, filtering the extractive, separating said substance from the filtrate with a reagent which acts as by adsorption, and separating the substance and reagent. h v

6. A method for obtaining apotent substance from the ductless portion of pancreatic or related glands in concentrated form and su'fiiciently free from impurities for repeated administration having the herein described physiological characteristicswhich consists of extracting said substance from the gland with a solvent, filtering the extractive, separatingsaid substance from the filtrate with a reagent .which acts as by adsorption, separating the substance and reagent, and making an aqueous sterile solution of said substance. I

7. A method for obtaining a potent substance from. the ductless portion of pancreatic or related glands in, concentrated form and sufliciently freefromimpurities for repeated administration having the hereindescribed physiological characteristics which consists of extracting said substance from the gland-,with a solvent, concentrating'said substance with benzoic acid, and sepgrating the substance from the benaoic aci ing the hereindescribed ph siologicalchar said " for repeated administration to patients hav- 8. A method for obtaining a potent substance from the ductless portion of pancreatic or related glands in concentrated form and sufliclently free from lmpurltles for repeated adminisrtation having the hereindescribed physiological characteristics winch consists of extracting said substance from the gland with a solvent, concentrating said substance with benzoic acid, separating the substance from the benzoic acid, and 7 form and sufficiently free from' impurities making an aqueous sterile solution 'of said substance. I I .t

9. A method for obtaining a potent-substancey-from' the ductless portion of pan-'- creatic or related glands in concentrated form and sufliciently free from impurities patients hav- I for repeated administration to ing the hereindescribed ph sio ogical characteristics whichconsists o extracting said substance from the gland with an acid sol- 35 vent and concentrating it with a reagent which will unite with it as by adsorption.-

10. A method for obtaining a potent substance from the ductless portion of pancreatic or related glands ,in concentrated form and sufiiciently free from impurities for re-. peated administration to patients having the hereindescribed ph siological character istics which consists 'o extractingsaid substance from the gland with a solvent, se arating the substance from the solution wit a reagent as a solid, dissolving said solid, and concentrating said substance with a reagent which will unite with it as by adsorption.

- 11. A method for obtaining a potent substance from the ductless portion of pan-' creatic or related glands in concentrated form and sufficiently free from" impurities for repeated administration to patients ha'vacteristics which consists o extractin substance from the gland with a so vent, separating the substance from-the solution and with ammonium sulphate, dissolving the,

ammonium sulphate from said substance,

and concentrating ,said substance with a re-, agent which will unite with it as by adsorp-' tion. I 5 w 12. method for obtaining a potent substance from-the ductless portion of pancreatic or related glands in concentrated form] and sufiiciently free from impurities for repeatedv administration to patients having the hereindescribed physiological, characteristics which consists o extracting said sub- 7 stance from the gland with a solvent separating the substance'from the solution with ammonium sulphate-dissolving the ammon-- iu'msulphate from said substance and con: 4

centrating said substance with benzo-ic aci 13. A method for obtaining a potent substance from the ductless .portion of pancreatic or related glands in concentrated form and sufliciently free from impurities lit) stance from the ductless portion of pnn' ing the hereindesci'ihed physiological chennctei'istics which consists of, extracting snid substance from the gland with e solvent sepcrating the substance from the solution with ammonium sulphate, dissolving the monium sulphate, from said substance, concenti'eting said substance with benzoic acid, and sepnmting sni'd substance from the henzoic ecid. 1

M. A method for obtaining a potent suboi'eotic on related glands in concentrated fomiend sntiiciently free from impurities toe repeated edminstintion to. atients hoving the hereindescribed physio ogicnl choirecteristics whichuconsists of extracting said substance from the gland with'oi? solvent,

se pemting the substance from the solution with ammonium sulphate, dissolving the emmonium sulphate from said substance concentreting snid suhstnnce with benzoic acid, sepomting' snid substance from the henzoic ecid, endmnhing an aqueous sterile solution of sn'id snhstnnce.

Dated at the said city of Toronto, this tenth dey of Jenunxy, A. D. 1923. 7 c

PETER JOSEPH MOLQNEY. DNALD M. FINDLAY.

" Witnesses: i

ldonnm McCmNToon,

STANLEY Rionnnns;

Patentedot. I

UNITE-D; s.r-A'n: s,rAr-1.-mr orrlca;

ra'rnasosnrn 'uonoimx AND normal: 14:. rmntax, or roiaoizro, onranro, caisson, assrouoas-rro 'rnn eovmmoas or THE UNIVERSITY or 'roaonro, or 'r'oiwmo, on'ranio, cmana. I 7

METHOD or rename AN nxrnAcn'omAmABLE raou THE MAMMALIAIJ' rmcnms on rnou 'rnn RELATED eranns or FISHES, USEFUL IN THE TREATMENT Y or DIABETES Matures.

m Drawing. Application manual- 1a, was. 8eral1Io.612,1 57. a. c

To all whom it may concern:

Be it known that we, PETER Josarn Moassociated substances. As extracting re- Lonny and DONALD MUNRO FINDLAY, British agents we found that boiling water produced sub ects, residing at the citvof- Toronto, in satisfactory results both in re ard ,-to the the county of ork and Province of Onmaximum yield of the potent su stance and tario,-Dominion of'Canada, have invented the small amount, of proteins and other 65 a new and useful method of preparing an gland substances in the extractive solution extract, obtainable from the mammalian and that copper sulphate solution pi'oduced' pancreas or from the related glands of fishes, substantially the same results as ,the boil;

l0 useful in the treatment of diabetes mellitus; ingvwater. i

.and we hereby declare that the following is e also found that-various or anic and so a full, clear, and exact description of thefinorganic reagents will-separate t e potent same. substance 'from the :other extractive sub- Investigations conducted by Banting,' stances and of these benzoic acid, formed 16 Best, and Colliprat the University of Toby adding sodiumbenzoate and hydrochloric will precipitate it free from adherent and .ronto proved that it is possible to obtain a acid. to the extractive solution will unite to,

potent substance or extract, containing inwith the potent substanee as by adsorption. concentrated form the secretion 01' hormone In an alternative method we found that from the ductless'portion of the pancreatic by adding an equal volume of saturated so- 20 glands of mammalia, and cartilaginous lution of ammoniunrsulphate to the aqueous fishes and the related lands (principal extractive solution, a solid separated out of m islets) of bony" fishes, su ciently free from the solution and floated to the top. This injurious substances for repeated adminissolid contained the potent substance and tration to human patients and having the some of the other extractive substances. We 20 physiological characteristics of removing removed thesolid and after eliminating the the cardinal objective s ptoms of diabetes adhering ammonium sulphate we dissolved 7 mellitus in patlentsan reducing the blood the solid in distilled water and separately su er in laboratory animals; added to the resulting solution sodium he methods employed during these inbenzoate and hydrochloricacid which vestigations involved the use of such v-reformed a precipitate of. benzoic acid and this agents as ethyl alcohol, methyl alcohol, precipitate united with the potent substance- 30 methylated spirits, and acetone, for extractas by adsorption ingt e secretion or hormone from the-gland" The :following is an example of. .one and then fractionally precipitating the pomethod by which'we obtained the potent as tent substance free from the associatedgland substance or: extract containing inconoentissue and the injurious substances in the treated form the internal secretion orhorextractive solution. V mone of the pancreas-of mammalia. p i From the resultsof our experience dur- I The fresh ancreaswas minced and mixed ing these investigations we concluded that. with an equa volume of boiling water which 40 it is possible to extract .the secretion or extracted from the gland the potent 'su'b hormone from the gland by various reagents stance derived from the secretion "or horwhich tend to retard or inhibitthe diges- 'mone. The temperatureot, the" resulting 'tive action of. the gland tissue and remove aqueous extractivesolution was maintains the injurioussubstances going into the exatfrom 85 C; to 100". C."for-a period of 45 tractive solution, and'that it is also possible. about five milliliter-iand then suddenly reto concentrate and purify the potent subducedto about 15 .C. at which it wasalstance in the extractive solution by the use lowedto stand. The boiling water tendedf of reagents which will unite with-it as-bfi to preventthe proteins 'goinghinto theexa adsorption orby theuse of reagentsjwhie tractive aqueoussolution wi the potent" T- I I I t I substance and .also tended to destroy was stirred or -agitated stand so that any inert material could settle,

enzymes, including trypsin, erepsin, and the l poteases.

When cooled the ids were rdmoved and the mixture was "filtered. 10 c. c. of

free it rem any of the filtrate from which it had been precipitated. The washed pre-' cipitate was then dried and the resulting product was a powder containing the potent substance or extract practically free from injurious substances; Although kept fora considerable time the'potency of the substance in thisform did not appre iably de-v teriorate under proper condit1o of sterility and temperature. A I

200 c. c. of 80% alcohol were added to the washed precipitate to dissolve the benzoic acid and potent substance and this solution after which it was ,ifiltered. After filtration i Berhfeldt filter, and contained the potent -substance. I

the major portion of the alcohol was boiled ofi from the filtrate which was 'thenshaken with ether and water and'allowed to stand, when two layers were manifested. The lower aqueous layer .contained the potent sub-- stanm in concentrated and purified form and the upper layer contained the benzoic acid. The loweraqueous layer was separated from the mixture and washed with ether to remove any remaining traces of alcohol and benzoicacid after-which the traces-of ether wereboiled ofi. The agueousresidue was then sterilized by filtration through a extracted the secretionor hormone" from. .the minced pancreas with ethyl alcohol, methyl alcohol, methylated spirits, and" aoetone,-as in'the methods of Banting, Best and Golliip and then concentrated -'the potent-s11 ance in the extractive filtratoby precipitating it with benzoic acid as above.

The following isanexample of the alter native method by'which we obtained the potent substance from the -pancreas of memmalia:

One kilogram of mixed with c; c. o 10% copper sulphate solution and 15c. cQOf'concentrated hydrochloric acid to retard the extrac ion ofthe proteins and other injurious sub ances from the gland. Two litres of water, heated to We also hydrochloric acid and then allowed to' off. The aqueous residue was then 'creatic or related glands in fground pancreas was 0., were then added to the mixture for extracting the potent substance. The re: sultin aqueous extractive mlu'tion was then filtere and to the filtrate was added an equal volume of saturated solution of ammonium sulphate. When the ammonium sulphate was added, a solid se arated out of the solution and floated to the top. -This soid contained the potent substance and some of e the other extractive substances. This solid was removed from the filtrate and freed from the adhering amanonium sulphate solution. The solid was then dissolved in was separately added sodium benzoate and which formed a precipitate of benzoic acid. This precipitate unit-' ed with the potent substance as by adsorption.

The benzoic acid precipitate with the ad- .hering potent substance was then separated from .the aqueous solution by filtering or by centrifuging and when separated was washed with a saturated aqueous solution of henzoic' acid to free it from any ammonium sulphate solution still; adhering to it. alcohol was thenadded to the washed pre-'. cipitate to dissolve, the benzoic acid. endpotent substance and make a concentration of 80% alcohol. This alcohol solution was stirred or agitated and c. c. oil-distilled water to which in suificient quantity to i then allowed to stand so that; any inert material could settle after which it was filtered. Afterfiltnation the major portion of the alcohol was boiled ofi from the filtrate which was then shaken with ether and water and allowed to stand when twolayers were manifested. The lower aqueous layer contained the potent substance in concentrated and purified form and the upperremainin tracesofalcohol and benzoic acid" after which the traces ofether; were boiled by filtration through a Berkfeldt filter and contained the potent substance. Havjngthus fully described our invention, wha we claim as new and desire to secure by tters,Patent.is:-- a l. A methodfor obtaining a potent substance fromtheductless portion of panconcentrated form and suiiiciently free from impurities for repeated administration to atients having the herein-described, physio ogical characteristics which consists of extracting said substance from thegland with a solvent and concentrating itwith a reagent which will unite with it as by adsorption. V I

2. A method for obtaining a potent sublayer contained the benzoicacid. The lower lllt) sterilized stance from the ductless portion ofcreatic or related glands-in concentrated 

